Apoptosis is a regulated process important for differentiation, control of cell number and removal of damaged cells. Failure to regulate apoptosis is a common feature in several diseases, including autoimmune disorders, neurodegenerative diseases and cancer. Apoptosis occurs through the activation of a cell-intrinsic suicide programme and is carried out by internal as well as external signals. The process of apoptosis can be divided in various phases that, at the end, activate signals leading to cell destruction. Although apoptosis is induced by a wide range of death stimuli, the execution phase of apoptosis is carried out, among others, by the caspases, that cleave target proteins leading to cell morphological changes.
Phosphorylation plays a critical role in the regulation of cell physiology and dysregulation of the mechanisms contributes to many disease states. Although much is known regarding alterations in kinase function in diseases such as cancer, the role of specific phosphatases in these same processes remains less characterized. Serine/threonine phosphatases are usually classified as type 1 (PP1) or type 2 (PP2), depending on their substrate specificity and sensitivity to inhibitors. PP1 represents a family of holoenzymes generated by specific interactions between catalytic subunits and a wide variety of regulatory or targeting subunits. PP1 is a major eukaryotic phosphatase that regulates diverse cellular processes such as cell cycle progression, proliferation, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, cytokinesis and neuronal signalling. During cell cycle, PP1 activity is regulated by phosphorylation. PP1 plays a key role in the mitotic transition by dephosphorylating proteins that are essential in these cellular functions. It has been shown that phosphorylation of PP1α at threonine 320 by cyclin-dependent kinases inhibits its enzymatic activity. In agreement, a constitutive mutant of PP1α that is resistant to cdk phosphorylation prevents cells from entering the S phase of cell cycle. Furthermore it was shown that IL-2 deprivation-induced apoptosis operates by regulating Bad dephosphorylation through the PP1α phosphatase (Ayllon et al., 2000) and that PP1α associates to caspase-9 to induce its dephosphorylation and, as a consequence, its protease activity (Dessauge et al., 2006).
Serine/threonine protein phosphatase 2A (PP2A) refers to a large family of dimeric or trimeric enzymes. The PP2A core enzyme consists of a catalytic C subunit (PP2Ac) and a structural A subunit. A third subunit (B) eventually binds to the core and these B subunits regulate both the substrate specificity and localization of PP2A holoenzymes. The A subunit primary serves a structural role and single amino acid alterations disrupt the binding of specific B subunits, suggesting that the A subunit regulates PP2A holoenzyme composition. Various PP2A complexes have been implicated in the control of a variety of cellular processes, including cell proliferation, survival, adhesion, cytoskeletal dynamics and malignant transformation.
A role of PP2A in apoptosis is suggested by its interaction with caspase-3, Bcl-2 and adenovirus E4orf4 protein. The activity of Bcl-2, an anti-apoptotic protein, is regulated by phosphorylation on Ser70, which is required for its anti-apoptotic role and can be reversed by PP2A. Moreover, IL-3 or bryostatin-1-induced phosphorylation of Bcl-2 on Ser70 is followed by increased association between Bcl-2 and PP2A prior to dephosphorylation of Bcl-2. Finally, PP2A regulates apoptosis via modulation of apoptotic signals such as NFkB, ERK and PI3K signalling pathways.
Penetrating peptides interacting with PP1/PP2A proteins were designed. This approach, named “Drug Phosphatase Technology” (DPT), was described in Guergnon et al, 2006 and International patent applications WO2003/011898 and WO2004/011595.